Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/713—Double-stranded nucleic acids or oligonucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/10—Cells modified by introduction of foreign genetic material
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/515—Complete light chain, i.e. VL + CL
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/50—Fusion polypeptide containing protease site
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
  • C07K2319/73—Fusion polypeptide containing domain for protein-protein interaction containing coiled-coiled motif (leucine zippers)

Definitions

• Antibody-based therapies have proven effective treatments for several diseases but in some cases, toxicities due to broad target expression have limited their therapeutic effectiveness. In addition, antibody-based therapeutics have exhibited other limitations such as rapid clearance from the circulation following administration.
• prodrugs of an active chemical entity are administered in a relatively inactive (or significantly less active) form. Once administered, the prodrug is metabolized in vivo into the active compound.
• prodrug strategies can provide for increased selectivity of the drug for its intended target and for a reduction of ad verse effects.
• antibodies, bi specific antibodies, activatable antibodies, and bi specific activatable antibodies are provided herein. These find use in therapeutics and diagnostics.
• the activatable antibodies and bispecific activatable antibodies of the present disclosure may be used to reduce damage to healthy tissue generally caused by an antibody binding to its target on healthy tissue as well as on diseased tissue.
• an activatable antibody comprising;
• VL light chain variable region
• VH heavy chain variable region
• MM masking moiety
• CM cleavable moiety
• the AB binds to CD3s.
• an aetivatable antibody comprising:
• a masking moiety 7 coupled to the AB, wherein the MM reduces or inhibits the binding of the AB to the CD3s when the AA is in an uncleaved state, wherein the MM comprises the amino acid sequence SEQ ID NO: 105 or SEQ ID NO: 106 or SEQ ID NO: 107;
• CM cleavable moiety
• a bispecific aetivatable antibody comprising the following structure:
• an IgG antibody (AB i) that specifically binds to a first target wherein the AB1 comprises:
• the MM1 reduces the binding of the AB1 to its target
• CM1 is a polypeptide that functions as a substrate for a first protease, b) two scFvs (AB2) that each specifically binds CD3c, wherein each AB2 comprises: (i) a light chain variable region (VL) linked to heavy chain variable region (Vi 1). wherein the carboxyl terminus of each AB2 is linked to the amino terminus of each of the AB1 heavy chains, wherein
• VL comprises an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 4 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 3;
• VL is linked to the VH by a linker L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 98 and SEQ ID NO: 108; and
• the MAC reduces the binding of the AB2 to CD3E
• the MAC comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 105, SEQ ID NO: 106 and SEQ ID NO: 107;
• CM2 is a polypeptide that functions as a substrate for a second protease.
• compositions comprising the AAs or BAAs, and, optionally, a carrier.
• a method of treating, alleviating a symptom of, or delaying the progression of a disorder or disease compri sing administering a therapeutically effective amount of the AAs or BAAs or of a pharmaceutical composition comprising an effective amount of the AAs or B AAs.
• any of the AAs, BAAs, or pharmaceutical compositions of the present disclosure for use as a medicament.
• an AA, a BAA, or a phar aceutical composition of the present disclosure for use in a method of treatment, optionally wherein the method is for the treatment or prevention of a neoplasm, a cancer, or a tumor.
• the cancer may be bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, sarcoma, squamous cell cancer, or skin cancer.
• the cancer may be associated with cells expressing EGFR, for instance the tumor cells may comprise cells expressing EGER or the j tumor microenvironment may comprise ceils expressing EGFR.
• provided herein is an AA, a B AA, or a pharmaceutical composition of the present disclosure for use in a method of treating a disorder or disease comprising disease cells expressing EGFR.
• provided herein is an AA, a BAA, or a pharmaceutical composition of the present disclosure for use in a method of treatment, wherein the treatment comprises the inhibition of angiogenesis.
• FIG. 1 demonstrates concentration-dependent aggregation of the dually masked bi specific activatable antibodies of Table 11, as assessed by studying the relation between monomer content and concentration.
• FIG. 2A demonstrates binding of intact (not activated) and activated BAAs CF138, 0139, and Cl 140 compared to 0106 as assessed by CD3s-based ELISA
• FIG. 2B demonstrates binding of intact and activated BAAs 0138, 0139, and 0140 compared to 0106, as assessed by EGFR-based ELISA.
• FIG. 3 demonstrates biological activity of intact and activated BAAs 0138, 0139, and 0140 compared to 0106, as assessed by cytotoxicity assays.
• FIG. 4 illustrates an exemplary BAA provided herein.
• FIG. 5 which plots tumor volume versus days post initial treatment dose, demonstrates a dose-dependent effect of 0106 and 0139 dually masked, bi specific, activatable antibodies on the growth of HT29-luc2 xenograft tumors. The most efficacious dose tested was 3.0 mg/kg, which led to tumor regression.
• FIG. 6 demonstrates the binding of 0106 and CI139 to HT29 and Jurkat cells via a flow ' cytometry-based binding assay compared to the activated bispecific antibody
• FIGS 7A-7C demonstrate biological activity of intact and activated BAA CI139 compared to Cl 106, as assessed by cytotoxicity assays in different cell lines.
• FIG. 8 which plots tumor volume versus days post initial treatment dose, demonstrates a dose-dependent effect of 0106 and 0139 dually masked, bispecific, activatable antibodies on the growth of HCT116 xenograft tumors.
• FIG. 9 demonstrates the pharmacokinetics in cynomolgus monkey of the dually masked molecules 0106 and 0139.
• AAs activatable antibodies
• BAAs bispecific antibodies
• AAs activatable antibodies
• BAAs bispecific activatable antibodies
• provided herein are humanized antibodies that specifically bind to the epsilon chain of CD 3 (CD3s; referred to herein interchangeably as CD3).
• CD3s epsilon chain of CD 3
• scFv antibodies that specifically bind to CD3.
• IgG antibodies that specifically bind to CD3.
• the anti-CD3 antibodies e.g., anti-CD3 scFv or IgG, such as IgG I, antibodies
• IgG antibodies that specifically bind to Epidermal Growth Factor Receptor (EGFR).
• IgGl antibodies that specifically bind to EGFR, wherein the antibodies comprise point mutations in the Fc region, such that the antibody has reduced effector function.
• AAs for example AAs that specifically bind to EGFR or CD3. These AAs are optimized for affinity, effector function, masking, and cleavability.
• BAAs for example BAAs that bind to a target antigen (e.g. tumor antigen, such as a target presented in Table 9) and a second antigen (e.g. immune effector antigen on an immune effector cell).
• a target antigen e.g. tumor antigen, such as a target presented in Table 9
• a second antigen e.g. immune effector antigen on an immune effector cell.
• the immune effector cell is a leukocyte cell.
• the immune effector cell is a T cell.
• the immune effector cell is a natural killer (Nik) cell.
• the immune effector cell is a macrophage.
• the immune effector cell is a mononuclear cell, such as a myeloid mononuclear cell.
• the BAAs are immune effector cell-engaging BAAs. In some embodiments, the BAAs are leukocyte cell-engaging BAAs. In some embodiments, the BAAs are T cell engaging bispecific (TCB) AAs. In some embodiments, the BAAs are NK cell-engaging BAAs. In some embodiments, the BAAs are macrophage cell-engaging BAAs. In some embodiments, the BAAs are mononuclear cell-engaging BAAs, such as myeloid
• the BAAs bind EGFR and CD3. These BAAs are optimized for affinity, effector function, masking, and cleavability.
• MMs masking moieties
• CMs eleavahie moieties
• the term“antibody” includes an antibody or antigen-binding fragment thereof that specifically binds its target and is a monoclonal antibody, domain antibody, single chain. Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.
• the antibody is an IgG antibody.
• the antibody is an IgGl antibody.
• the antibody is an IgG4 antibody.
• the antibody is a scFv antibody.
• such an antibody or immunologicalJy active fragment thereof that binds its target is a mouse, chimeric, humanized or fully human monoclonal antibody.
• CD3B an antibody or antigen binding fragment thereof (AB) that specifically binds to the epsilon chain of CDS
• CD3B epsilon chain of CDS
• AAs activatable antibodies
• BAAs bispecific activatabie antibodies
• Exemplar ⁇ ' amino acid sequences of CD3-binding antibodies of the disclosure are provided in Table 1. (Predicted CDR sequences are underlined). As provided below, L3 is a linker, linking the light and heavy chain variable domains, in the exemplary CD3 -binding antibodies. [0034] Exemplar ⁇ - scFv linkers (referred to herein as“L3” linking a YL and VH) are provided in Table 1-1.
• the CD3 antibody comprises at least one, at least two, ast least three, at least four, or at least five of the CDR sequences provided in Table 2 In some embodiments, the CD3 antibody comprises the six CDR sequences provided in Table 2.
• the CD3 antibody comprises heavy chain variable domain as set forth in SEQ ID NO: 2.
• the CD3 antibody comprises a heavy chain variable domain as set forth in SEQ ID NO: 3.
• the CD3 antibody comprises a light chain variable domain as set forth in SEQ ID NO: 1.
• the CDS antibody comprises a light chain variable domain as set forth in SEQ ID NO: 4
• the CD3 antibody comprises a heavy chain variable domain as set forth in SEQ ID NO: 2 and a light chain variable domain as set forth in SEQ ID NO: 1 [0042] In some embodiments, the CD3 antibody comprises a heavy chain variable domain as set forth in SEQ ID NO: 3 and a light chain variable domain as set forth in SEQ ID NO: 1.
• the CD3 antibody comprises a heavy chain variable domain as set forth in SEQ ID NO: 3 and a light chain variable domain as set forth in SEQ ID NO: 4 [0044] In some embodiments, the CD3 antibody comprises a heavy chain variable domain as set forth in SEQ ID NO: 2 and a light chain variable domain as set forth in SEQ ID NO: 4.
• the CD3 antibody comprises a heavy chain variable domain as set forth in SEQ ID NO: 2 or SEQ ID NO: 3 or comprises a light chain variable domain as set forth in SEQ ID NO: I or SEQ ID NO: 4.
• the CDS antibody comprises a heavy chain variable domain as set forth in SEQ ID NO: 2 or SEQ ID NO: 3 and comprises a light chain variable domain as set forth in SEQ ID NO: 1 or SEQ ID NO: 4.
• the antibody comprises a sequence 70%, 80%, 90%, 95%, 99%, or 100% similar to a heavy chain variable domain as set out above, and/or comprises a sequence 70%, 80%, 90%, 95%, 99%, or 100% similar to a light chain variable domain as set out above, and specifically binds to CD3.
• heavy chain variable domain is in combination with a light chain variable domain as set out above.
• the antibody comprises a sequence 70%, 80%, 90%, 95%, 99%, or 100% similar to a heavy chain variable domain as set out above, and/or comprises a sequence 70%, 80%, 90%, 95%, 99%, or 100% simi lar to a light chain variable domain as set out above, comprises the six CDR sequences provided in Table 2, and specifically binds to CD3.
• the CD3 antibody is a scFv antibody.
• the variable domains comprise the following structure from N terminus to C terminus: LV— HV. In some embodiments, the variable domains comprise the following structure from N terminus to C terminus: HV— -LV.
• the CD3 antibody is a scFv antibody comprising a light chain variable region (VL) linked to a heavy chain variable region (VH), wherein the VL is linked to the VH by a linker comprising amino acid sequence SEQ ID NO: 98.
• a linker comprising amino acid sequence SEQ ID NO: 98.
• the CD3 antibody is a scFv antibody comprising a light chain variable region (VL) linked to a heavy chain variable region (VH), wherein the VL is linked to the VH by a linker comprising amino acid sequence SEQ ID NO: 108.
• VL light chain variable region
• VH heavy chain variable region
• Exemplary sequences with such a linker are provided in Table 1.
• an antibody that specifically binds to CD3 wherein the antibody is an IgGl antibody or a scFv linked to an Fc domain, wherein the antibody comprises an Fc region comprising an amino acid substitution in at least one of amino acid positions L234, L235, and P331, as numbered by the ELI index as set forth in Kabat, such that the antibody has reduced effector function.
• the amino acid substitution is any one or more of L234F, L235E, and P331S.
• the antibody comprises amino acid substitutions in at least two of amino acid positions L234, L235, and P331. In some embodiments, the antibody comprises amino acid substitutions at amino acid positions L234, L235, and P331. In some embodiments, the antibody comprises L234F, 1,23513, and P331 S amino acid substitutions. In some
• the antibody comprises an Fc region comprising an amino acid substitution at N297. In some embodiments, the Fc region comprises an N297Q mutation. In some embodiments, the antibody comprises L234F, L235E, P331S, and N297Q amino acid substitutions. In some embodiments, the heavy chain vari able domain of the antibody with reduced effector function comprises any one of SEQ ID NO: 2 or SEQ ID NO: 3 or wherein the light chain vari able domain of the AB comprises any one of SEQ ID NO: 1 or SEQ ID
• any one of the CD3 antibodies provided herein is in an activatable antibody (AA) format.
• the AAs of the disclosure comprise MM-CM constructs, also referred to herein as a prodomain.
• prodomain refers to a polypeptide comprising a masking moiety (MM) and a cleavable moiety (CM).
• the MM and the CM are separated by a linker, referred to herein as LI
• the prodomain comprises a linker at the carboxyl terminus of the CM; this linker, referred to herein as L2, links the CM of the prodomain to the AB.
• the prodomain comprises a linker between MM and CM and a linker after CM.
• a prodomain comprises one of the following formulae (where the formula below represents an amino acid sequence in either N- to C-terminal direction or C- to N-terminal direction): (MM)-Ll-(CM), (MM)-(CM)-L2, (MM)-L1-(CM)-L2, or (MM) ⁇ (CM).
• a prodomain comprises an EGFR MM and a CM cleavable by a matriptase or MMP; or a CD3E MM and a CM cleavable by a matriptase or MMP.
• a prodomain comprises an EGFR ADM and a CM that is cleavable by a matriptase and an MMP. In some embodiments, a prodomain comprises a CD3s MM and a CM! that is cleavable by a matriptase and an MMP.
• AAs activatable antibodies
• nucleotides encoding a prodomain of the disclosure.
• a CD3 AA comprising: (a) an antibody or antigen binding fragment thereof (AB) that specifically binds to the epsilon chain of CDS (CD3s), wherein the antibody comprises a heavy chain domain as set forth in SEQ ID NO: 2 or SEQ ID NO: 3 or comprises a light chain domain as set forth in SEC) ID NO: 1 or SEQ ID NO: 4; and (b) a prodomain, wherein the prodomain comprises a (i) masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits the binding of the AB to the CDSs when the AA is in an uncleaved state; and (ii) a cleavabie moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease.
• AB antibody or antigen binding fragment thereof
• a CD3 AA comprising: (a) an antibody or antigen binding fragment thereof (AB) that specifically binds to CD3s; and (b) a prodomain, wherein the prodomain comprises: (i) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits the binding of the AB to the CD3s when the AA is in an uncieaved state, wherein the MM comprises the amino acid sequence SEQ ID NO: 105 or SEQ ID NO: 106 or SEQ ID NO: 107; and (ii) a cleavabie moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease.
• CM cleavabie moiety
• a CD 3 AA comprising: (a) at least one scFv comprising a light chain variable region (VL) linked to a heavy chain variable region (VH), wherein the VL is linked to the VH by a linker comprising amino acid sequence SEQ ID NO: 108 or SEQ ID NO: 98; and (b) a prodomain comprising: (i) a masking moiety (MM) coupled to the scFv, wherein the MM reduces or inhibits the binding of the scFV to its target when the AA is in an uncleaved state; and (ii) a cleavabie moiety (CM) coupled to the scFv, wherein the CM is a polypeptide that functions as a substrate for a protease.
• MM masking moiety
• CM cleavabie moiety
• a CD 3 AA which comprises the following structure, when not activated: (a) an antibody or antigen binding fragment thereof (AB) that specifically binds to the epsilon chain of CD3 (CD3s) when activated, wherein the antibody compri ses a heavy chain domain 70%, 80%, 90%, 95%, 99%, or 100% similar to a sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 3 or comprises a light chain domain 70%, 80%, 90%, 95%, 99%, or 100% similar to a sequence as set forth in as set forth in SEQ ID NO: 1 or SEQ ID NO: 4; and (b) a prodomain, wherein the prodomain comprises a (i) masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits the binding of the AB to the CD3s when the AA is in an uncleaved state; and (ii) a cleavabie moiety (CM
• a CD3s AA when activated, specifically binds to a target
• said AA when not activated, comprises the following structure: (a) an antibody or antigen binding fragment thereof (AB) that specifically binds to CD3s; and (b) a prodomain comprising: (i) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits the binding of the AB to the CD3s when the AA is in an uncleaved state, wherein the MM comprises the amino acid sequence SEQ ID NO: 105 or SEQ ID NO: 106; or SEQ ID NO: 107 and (ii) a cleavable moiety ' (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease.
• CM cleavable moiety '
• a AA when activated, specifically binds to a target
• said AA when not activated, comprises the following structure: (a) an antibody or antigen binding fragment thereof (AB) that specifically binds to CD3e; and (b) a prodomain comprising: (i) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits the binding of the AB to the CD3s when the A A is in an un cleaved state, wherein the MM comprises the amino acid sequence SEQ ID NO: 105 or SEQ ID NO: 106; or SEQ ID NO: 107 and (ii) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease
• the AB of the CD3 AA is any one of the CD3 antibodies described in the preceding section
• the AB of the CD3 A A comprises a heavy chain variable domain as set forth in SEQ ID NO: 2
• the AB of the CD3 AA comprises a heavy chain variable domain as set forth in SEQ ID NO: 3.
• the AB of the CD3 AA comprises a light chain variable domain as set forth in SEQ ID NO: 1.
• the AB of the CDS AA compri ses a light chain variabl e domain as set forth in SEQ ID NO: 4
• the AB of the CD3 AA comprises a heavy chain variable domain as set forth in SEQ ID NO: 2 and a light chain domain as set forth in SEQ ID NO: 1.
• the AB of the CDS AA comprises a heavy chain variable domain as set forth in SEQ ID NO: 3 and a light chain domain as set forth in SEQ ID NO: 1.
• the AB of the CDS AA comprises a heavy chain variable domain as set forth in SEQ ID NO: 3 and a light chain domain as set forth in SEQ ID NO: 4
• the AB is a scFv comprising a light chain variable region (VL) linked to a heavy chain variable region (VH), wherein the VL is linked to the VH by a linker comprising amino acid sequence SEQ ID NO: 108. Exemplary sequences with such a linker are provided in Table 1.
• the AB is a scFv comprising a light chain variable region (VL) linked to a heavy chain variable region (VH), wherein the VL is linked to the VH by a linker comprising amino acid sequence SEQ ID NO: 98.
• a linker comprising amino acid sequence SEQ ID NO: 98.
• the MM of the CD3 A A comprises any one of the sequences set forth in Table 3.
• CDS masking moieties (MMs) of the disclosure are provided in Table 3.
• the MM of the CD3 AA comprises the sequence set forth in SEQ ID NO: 12. In some embodiments, the MM of the CDS AA is the sequence set forth in SEQ ID NO: 10. In some embodiments, the MM of the CD3 AA is the sequence set forth in SEQ ID NO: 11. In some embodiments, the MM of the CDS AA comprises the sequence set forth in SEQ ID NO: 105. In some embodiments, the MM of the CD3 AA comprises the sequence set forth in SEQ ID NO: 106. In some embodiments, the MM of the CD3 AA comprises the sequence set forth in SEQ ID NO: 107.
• the C : of the CDS AA comprises any one of the sequences set forth in Table 4.
• Exemplary cleavable moieties (CMs) of the disclosure are provided in Table 4.
• a cleavable moiety of the CD3 A A comprises any of the amino acid sequences selected fro the group consisting of SEQ ID NO: I3-!7.
• the CM of an AA of the disclosure comprises any one of the sequences set forth in Table 4-1.
• the CM of an AA of the disclosure comprises any of the amino acid sequences selected from the group consisting of SEQ ID NO: 18-56.
• IgGl antibodies that that have Fc mutations or antibody fragments containing antigen-binding domains (e.g. scFv, Fab, F(ab’)2) linked to a Fc domain, wherein the Fc exhibits reduced effector function (referred to herein as Fc variants).
• Fc variants e.g. scFv, Fab, F(ab’)2
• the antibodies that comprise these Fc mutations result in reduced effector function, while maintaining target binding affinity. Accordingly, provided herein are antibodies that bind to a target of interest, wherein the antibody is an IgGl antibody or an antibody fragment linked to an Fc, wherein the Fc region comprises an amino acid substitution in at least one of amino acid positions L234, 1235, and P331 , as numbered by the EU index as set forth in Kabat, such that the antibody has reduced effector function.
• the amino acid substitution is any one or more of L234F, L235E, and P331 S.
• the amino acid substitution is N297Q or N297A.
• the Fc is selected from the Fc sequences presented in Table 4-2.
• Antibodies, AAs, bispecific antibodies, and BAAs comprising these Fc mutations are provided herein
• the Fc domain of such antibodies, AAs, bispecific antibodies, and BAAs comprise any one of the sequences set forth in SEQ ID NO: 115, SEQ ID NO; 1 16, SEQ ID NO: 1 17, SEQ ID NO: 118, SEQ ID NO; 1 19, SEQ ID NO: 120, SEQ ID NO: 121 and SEQ ID NO: 122, as set forth in Table 4-2.
• such Fc variant-containing AAs and BAAs can bind an immune effector cell. In some embodiments, they can bind a target selectively located on an immune effector cell. In some embodiments, they can bind CD3. In some embodiments, they can bind any target listed in Table 9. In some embodiments, they can bind EGFR.
• an activatable antibody comprising: an antibody (AB) that specifically binds a target, wherein the antibody is an IgGl antibody, and wherein the Fc region of the antibody comprises an amino acid substitution in at least one of amino acid positions L234, 1,235, and P331 , as numbered by the EU index as set forth in Rabat, such that the AA has reduced effector function; and a prodomain, wherein the prodomain comprises:
• MM masking moiety
• CM cleavable moiety
• an activatable antibody comprising: an antibody (AB) that specifically binds a target, wherein the antibody is an IgGl antibody, and wherein the Fc region of the antibody comprises an amino acid substitution in at least one of amino acid positions L234, L235, and P331, as numbered by the EU index as set forth in Rabat, such that the AA has reduced effector function; and a prodomain, wherein the prodomain comprises:
• MM masking moiety
• CM cleavable moiety
• the Fc region comprises amino acid substitutions in at least amino acid positions L234, L235, N297 and P331 (e.g. L234F, L235E, P331S, and/or N297A or N297Q), as numbered by the EU index as set forth in Kabat, such that the AA has reduced effector function.
• the target is selected from the group consisting of the targets presented in Table 9.
• a bispecific activatable antibody comprising:
• an IgG antibody that specifically binds to a first target wherein the AB1 comprises:
• two first prodomains each comprising a first masking moiety (MM!) linked to a first cleavab!e moiety (CM1) in the N-terminal to C-terminal direction, wherein the carboxyl terminus of each first prodomain is linked to the amino terminus of each light chain of the ABI, wherein the MM1 reduces or inhibits the binding of the ABI to its target; and the CM1 is a polypeptide that functions as a substrate for a first protease,
• each AB2 comprises:
• VL light chain variable region
• VH heavy chain variable region
• the VL comprises an amino acid sequence as set forth in SEQ ID NO: l or SEQ ID NO: 4
• the VH comprises an amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 3
• the VL is linked to the VH by a linker (L3) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 98 and SEQ ID NO: 108;
• two second prodomains each comprising a second masking moiety (MM2) linked to a second cleavable moiety (CM2), in the N-terminal to C-terminal direction, wherein the carboxyl terminus of each second prodomain is linked to the amino terminus of each AB2 wherein the MM2 reduces the binding of the AB2 to CD3s;
• the MM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 105, SEQ ID NO: 106 and SEQ ID NO: 107;
• the CM2 is a polypeptide that functions as a substrate for a second protease.
• AB antigen binding fragments thereof
• EGFR antibodies bispecific antibodies targeting EGFR, AAs capable of binding EGFR upon activation, and BAAs capable of binding EGFR upon activation.
• the EGFR antibody comprises the CDRs of Table 5.
• IgGl antibodies that specifically bind to the Epidermal Growth Factor Receptor (EGFR) and impart reduced effector function.
• the antibodies comprise Fc mutations that result in reduced effector function, while maintaining EGFR binding affinity.
• antibodies that bind to EGFR wherein the antibody is an IgGl antibody, wherein the antibody comprises an Fc region comprising an amino acid substitution in at least one of amino acid positions L234, L235, and P331, as numbered by the EU index as set forth in Rabat, such that the antibody has reduced effector function.
• the amino acid substitution is any one or more of L234F, L235E, and P331S.
• the antibody comprises amino acid substitutions in at least two of amino acid positions L234, L235, and P331.
• the antibody comprises amino acid substitutions at amino acid positions L234, 1235, and P331.
• the antibody comprises L234F, L235E, and P331S amino acid substitutions.
• the antibody comprises an Fc region comprising an amino acid substitution at N297 along with an amino acid substitution in at least one of amino acid positions L234, L235, and/or P33 I .
• the Fc region comprises an N297Q mutation.
• the Fc region comprises an N297A mutation.
• the antibody comprises L234F, L235E, P331S and N297Q substitutions. In some embodiments, the antibody comprises L234F, L235E, P331S and N297A substitutions
• exemplary amino acid sequences of EGFR-binding antibodies are provided in Table 6.
• (YL and VH denote the variable light and variable heavy chains, respectively;
• LC and HC denote the light and heavy chains, respectively).
• the EGFR antibodies comprise any one of the sequences provided in Table 6.
• the heavy chain of the EGFR antibody comprises any one of the sequences set forth in SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO:
• the notation Fcnrt3 comprises a triple point mutation, wherein the Fc region of the heavy chain of the EGFR antibody comprises the following three point mutations: L234F, L235E, and P331S. Accordingly, in some embodiments, the EGFR antibody comprises a heavy chain with an amino acid sequence set forth in SEQ ID NO: C225v5Fcmt3 HC. In some embodiments, the Fc region of the heavy chain of the EGFR antibody comprises a fourth point mutation, N297Q. The notation Fcmt4 comprises the Fcmt3 triple point mutation and the fourth point mutation, N297Q. Accordingly, in such embodiments, the EGFR antibody comprises a heavy chain with an amino acid sequence set forth in SEQ ID NO: 76.
• the AB comprises a sequence 70%, 80%, 90%, 95%, 99%, or 100% similar to a heavy chain variable domain set out in SEQ ID NO: 64, and/or comprises a sequence 70%, 80%, 90%, 95%, 99%, or 100% similar to a light chain variable domain as set out in SEQ ID NO: 63, and specifically binds to EGFR.
• the AB comprises a sequence 70%, 80%, 90%, 95%, 99%, or 100% similar to a heavy chain variable domain as set out in SEQ ID NO: 64, and/or comprises a sequence 70%, 80%, 90%, 95%, 99%, or 100% similar to a light chain variable domain as set out in SEQ ID NO: 63, comprises the six CDR sequences provided in Table 5, and specifically binds to EGFR.
• the AB comprises an Fc region as set out above or in Table 6.
• any one of the EGFR antibodies provided herein are in an AA format (EGFR AAs). Accordingly provided herein are AAs comprising antibodies or antigen binding fragments thereof (AB) that specifically bind to EGFR Exemplary CDR sequences of EGFR-binding antibodies are provided in Table 5.
• the AA comprises: (a) any antibody or an antigen binding fragment thereof (AB) that specifically binds to Epidermal Growth Factor Receptor (EGFR); and (b) a prodomain, wherein the prodomain comprises (i) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits the binding of the AB to the EGFR when the
• AA is in an uncleaved state, and wherein the MM comprises an amino acid sequence selected from the group consisting of sequences presented in Table 7; and (ii) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease.
• CM cleavable moiety
• Exemplar ⁇ ' EGFR masking moieties (MMs) of the disclosure are provided in Table 7 and Table 8.
• the MM of the EGFR AA comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the MM of the EGFR AA comprises the amino acid sequence of SEQ ID NO: 85.
• the CM of the EGFR AA comprises an amino acid sequence selected from the group consisting of sequences presented in Table 4. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO: 16.
• an activatable antibody comprising: (a) an antibody that specifically binds to Epidermal Growth Factor Receptor (EGFR), wherein the antibody is an IgGl antibody, and wherein the Fc region of the antibody comprises an amino acid substitution in at least one of amino acid positions L234, L235, and P331, as numbered by the EU index as set forth in Rabat, such that the AA has reduced effector function; (b) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits the binding of the AB to the EGFR when the AA is in an uncleaved state; and (c) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease.
• EGFR IgGl antibodies can be any of the IgGl antibodies described in the immediately preceding section.
• the MM comprises an amino acid sequence selected from
• an activatable antibody comprising: (a) an antibody (AB) that specifically binds to Epidermal Growth Factor
• EGFR EGFR Receptor
• the AB is an IgGl antibody
• the Fc region of the AB comprises an amino acid substitution in at least one of amino acid positions L234, 1,235, and P331, as numbered by the EU index as set forth in Rabat, such that the AA has reduced effector function
• a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits the binding of the AB to the EGFR when the AA is in an un cleaved state
• CM c!eavable moiety
• the amino acid substitution is any one or more of L234F, L235E, and P331S.
• the AB comprises amino acid substitutions in at least two of amino acid positions L234, L235, and P331. In some embodiments, the AB comprises amino acid substitutions at amino acid positions L234,
• the AB comprises L234F, L235E, and P331S amino acid substitutions.
• the AB comprises an Fc region comprising an amino acid substitution at N297.
• the Fc region comprises an N297Q mutation.
• the AB comprises L234F, L235E, P331S, and N297Q amino acid substitutions.
• the MM comprises an amino acid sequence selected from the group consisting of sequences presented in Table 7 or Table 8. In some embodiments, the MM comprises the amino acid sequence of SEQ ID NO: 78.
• the MM comprises the amino acid sequence of SEQ ID NO: 85
• the CM comprises an amino acid sequence selected from the group consisting of sequences presented in Table 4.
• the CM comprises the amino acid sequence of SEQ ID NO: 14.
• the CM comprises the amino acid sequence of SEQ ID NO: 16.
• the AA is part of a BAA.
• BAAs Bisperifie. Activatable Antibodies
• BAAs bispecific AAs, BAAs
• said bi specific AA when activated, specifically binds to two targets (e.g. binds two different targets, or binds two different epitopes on the same target) and can comprise the exemplary structure provided in FIG. 17.
• the first target is selected from the group consisting of the targets presented in Table 9 and the second target is selected from the group consisting of the targets presented in Table 9.
• the BAAs of the disclosure comprise MM-CM constructs, also referred to herein as a prodomain.
• prodomain refers to a polypeptide comprising a masking moiety (MM) and a cleavable moiety (CM).
• MM masking moiety
• CM cleavable moiety
• the M : and the CM are separated by a linker, referred to herein as LI .
• LI linker
• the prodomain comprises a linker at the carboxyl terminus of the CM; this linker, referred to herein as L2, links the CM of the prodomain to the AB.
• the prodomain comprises a linker between MM and CM and a linker after CM.
• the MM and the CM are not separated by a linker.
• a prodomain comprises one of the following formulae (where the formula below represents an amino acid sequence in either N- to C-terminal direction or C- to N- termina! direction): (MM)-Ll-(CM), (MM)-(CM)-L2, (MM)-L1-(CM)-L2, or (MM)-(CM).
• a prodomain comprises an EGFR MM and a CM cleavable by a matriptase or MMP; or a CD3E MM and a CM cleavable by a matriptase or MMP.
• a prodomain comprises an EGFR MM and a CM that is cleavable by a matriptase and an MMP.
• a prodomain comprises a CD3E MM and a CM that is cleavable by a matriptase and an MMP.
• BAAs bispecific activatable antibodies
• a BAA wherein said BAA, when activated, specifically binds to two targets (e.g two different targets; or two different epitopes on the same target), and wherein said BAA, when not activated, comprises the following structure:
• an IgG antibody that specifically binds to a first target wherein the AB 1 comprises:
• AB1 HCs heavy chains
• AB1 LCs light chains
• first prodomains each comprising a first masking moiety (MM1) linked to a first cleavable moiety (CM1) in the N-terminal to C-terminal direction, wherein the carboxyl terminus of each first prodomain is linked to the amino terminus of each light chain of the AB1 , wherein
• VL light chain variable region
• VH heavy chain variable region
• the VL comprises an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 4
• the VH comprises an amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 3
• the VL is linked to the VH by a linker (L3) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 98 and SEQ ID NO: 108, and
• MM2 linked to a second cleavable moiety (CM2), in the N-terminal to C-terminal direction, wherein the carboxyl terminus of each second prodomain is linked to the amino terminus of each AB2 wherein
• the MM2 reduces the binding of the AB2 to CD3s
• the MM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12 , SEQ ID NO: 105 , SEQ ID NO: 106 and SEQ ID NO: 107 ; and
• the CM2 is a polypeptide that functions as a substrate for a second protease
• the VL is linked to the VH by a linker (L3) comprising amino acid sequence SEQ ID NO: 98
• the VI is linked to the VH by a linker (L3) comprising amino acid sequence SEQ ID NO: 108.
• the MM2 comprises amino acid sequence SEQ ID NO: 12.
• the MM2 comprises amino acid sequence SEQ ID NO: 105.
• the MM2 comprises amino acid sequence SEQ ID NO: 106
• the MM2 comprises amino acid sequence SEQ ID NO: 107.
• the AB1 binds a tumor target.
• the AB1 binds EGFR.
• the EGFR-binding AB1 may be any EGFR-binding antibody, or fragment thereof, as disclosed herein
• the AB2 may be any CD3-binding antibody, or fragment thereof, as disclosed herein.
• the MM1 comprises an amino acid sequence selected from the group consisting of sequences presented in Table 7 or Table 8.
• the MM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 78 and SEQ ID NO: 85.
• the MM I comprises amino acid sequence SEQ ID NO: 78.
• the MM1 comprises amino acid sequence SEQ ID NO: 85.
• the CM ! comprises the amino acid sequence of any one of the CMs listed in Table 4 or Table 4-1 [0123] In some embodiments, the CM2 comprises the amino acid sequence of any one of the CMs listed in Table 4 or Table 4-1
• the CM4 or CM2 comprises the amino acid sequence of any one of the CMs listed in Table 4 or Table 4-1
• the CM1 comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 17.
• the CM2 comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 17.
• the CM1 or CM2 comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 17.
• the CM! comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 16.
• the CM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 17
• the AB 1 comprises an Fc region comprising an amino acid substitution in at least one of amino acid positions L234, L235, N297, and P331, as numbered by the EU index as set forth in Kabat, such that the BAA has reduced effector function.
• the AB1 comprises amino acid substitutions in at least two of amino acid positions L234, L235, and P331.
• the AB 1 comprises amino acid substitutions at amino acid positions L234, L235, and P331.
• the AB1 comprises L234F, L235E, and P331 S amino acid substitutions.
• the AB 1 comprises an Fc region comprising an amino acid substitution at N297
• the AB1 comprises L234F, L235E, P331 S, and N297Q amino acid substitutions.
• the heavy chain of AB1 comprises an amino acid sequence selected from the group consisting SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, as set forth in Table 6.
• the BAA is CI138, comprising the layout and sequence as provided in Tables 1 1 and 1.
• the BAA is 0138, wherein the heavy chain sequence comprises an amino acid sequence selected from the group consisting SEQ ID NO: 155 or SEQ ID NO: 124 and the light chain sequence comprises an amino acid sequence selected from the group consisting SEQ ID NO: 132 or SEQ ID NO: 140.
• the BAA is 0139, comprising the layout and sequence as provided in Tables 11 and 1. .
• the BAA is 0139, wherein the heavy chain sequence comprises an amino acid sequence selected from the group consisting SEQ ID NO: 157 or SEQ ID NO:126 and the light chain sequence comprises an amino acid sequence selected from the group consisting SEQ ID NO: 132 or SEQ ID NO: 140.
• the BAA is 0158, comprising the layout and sequence as provided in Tables 11 and 1.
• the BAA is 0158, wherein the heavy chain sequence comprises an amino acid sequence selected from the group consisting SEQ ID NO: 161 or SEQ ID NO: 128 and the light chain sequence comprises an amino acid sequence selected from the group consisting SEQ ID NO: 132 or SEQ ID NO: 140.
• the BAA is 0140, comprising the layout and sequence as provided in Tables 1 1 and 1.
• the BAA is Cl 140, wherein the heavy- chain sequence comprises an amino acid sequence selected from the group consisting SEQ ID NO: 159 or SEQ ID NO:25 and the light chain sequence comprises an amino acid sequence selected from the group consisting SEQ ID NO: 132 or SEQ ID NO: 140.
• the BAAs provided herein comprise:
• an IgG antibody (AB 1) that specifically binds to a first target wherein the AB 1 comprises:
• MM1 first masking moiety- linked to a first cleavable moiety (CMl) to form a prodomain
• CMl first cleavable moiety
• the MM1 inhibits the binding of the ABl to its target
• CM! is a polypeptide that functions as a substrate for a first protease, d) two scFvs (AB2) that each specifically bind to a second target wherein each AB2 comprises:
• a heavy chain variable region linked to a light chain variable region wherein the carboxyl terminus of each AB2 is linked to the amino terminus each of the ABl heavy chains
• a second masking moiety (MM2) linked to a second cleavable moiety (CM2) to form a second prodomain wherein the carboxyl terminus of the second prodomain is linked to the amino terminus of each AB2 wherein the MM2 inhibits the binding of the AB2 to its target
• CM2 is a polypeptide that functions as a substrate for a second protease
• MM2 comprises amino acid sequence SEQ ID NO: 105 or SEQ ID NO:
• MM! comprises an amino acid sequence selected from the group consisting of sequences presented in Table 7,
• AB2 comprises a heavy chain variable domain as set forth in SEQ ID NO: 2 or SEQ ID NO: 3 or a light chain variable domain as set forth in SEQ ID NO: 1 or SEQ ID NO: 4;
• iv. AB1 comprises an Fc region comprising an amino acid substitution in at least one of amino acid positions L234, L235, N297, and P331 or L234, L235 and P331, as numbered by the EU index as set forth in Rabat, such that the BAA has reduced effector function.
• the BAAs provided herein comprise:
• an IgG antibody (AB1) that specifically binds to a first target wherein the AB1
• MM1 a first masking moiety linked to a first cleavable moiety (CM!) to form a prodomain, wherein the carboxyl terminus of the prodomain is linked to each amino terminus of each light chain of the ABI, wherein
• the MM I inhibits the binding of the ABI to its target
• CM1 is a polypeptide that functions as a substrate for a first protease
• each AB2 comprises:
• a heavy chain variable region linked to a light chain variable region, wherein the carboxyl terminus of each AB2 is linked to the amino terminus each of the AB 1 heavy chains; and b. a second masking moiety (MM2) linked to a second cleavable moiety (CM2) to form a prodomain, wherein the carboxyl terminus of the prodomain is linked to the amino terminus of each AB2 wherein
• the MM2 inhibits the binding of the AB2 to its target
• the CM2 is a polypeptide that functions as a substrate for a second protease
• the AB1 comprises an Fc region comprises an amino acid substitution in at least one of amino acid positions L234, L235, N297, and P331, as numbered by the EU index as set forth in Kabat, such that the BAA has reduced effector function.
• the Fc region comprises amino acid substitutions in at least two amino acid positions L234, L235, N297 and P331, as numbered by the EU index as set forth in Kabat, such that the BAA has reduced effector function.
• the Fc region comprises amino acid substitutions in at least amino acid positions L234, L235, and P331, as numbered by the EU index as set forth in Kabat, such that the BAA has reduced effector function.
• the first target is selected from the group consisting of the targets presented in Table 9 and the second target is selected from the group consisting of the targets presented in Table 9.
• AB1 binds a target antigen, e.g. a tumor antigen, and the AB2 binds an immune effector target.
• AB2 binds a target antigen, e.g. a tumor antigen, and the AB1 binds an immune effector target.
• the AB1 binds EGFR and the AB2 binds CD3.
• the AB1 is an EGFR-binding antibody, or fragment thereof, as disclosed herein
• the AB2 is a CD3-binding antibody, or fragment thereof, as disclosed herein.
• the MM1 comprises an amino acid sequence selected from SEQ ID NOs: 78-87. In some embodiments, the MM1 comprises SEQ ID NO: 78.
• the MM2 comprises an amino acid sequence selected from SEQ ID NOs: 12, 106, 107 and 105. In some embodiments, the MM2 comprises the amino acid sequence SEQ ID NO: 12.
• the bispecific AA is Cl 106, 0138, 0139, 0158 or 0140 as provided in Table 11, in Example 1.
• AB1 comprises the amino acid sequence of
• the first and second proteases are the same protease. In some embodiments, the first and second proteases are different proteases. In some embodiments, CM1 and CM2 comprise the same amino acid sequence. In some embodiments,
• CM1 and CM2 comprise different amino acid sequences.
• CM! and CM2 comprise different amino acid sequences that are cleavable by the same protease or proteases. In some embodiments, CM! and CM2 are cleavable by more than one protease. In some embodiments, CM1 and/or CM2 is cleavable by a serine protease. In some embodiments, CM! and/or CM2 is cleavable by a matrix metalloproteinase (MMP). In some embodiments, CM1 and/or CM2 is cleavable by a serine protease and an MMP
• MMP matrix metalloproteinase
• Exemplar ⁇ - BAAs of the disclosure include, for example, those shown in the
• At least one of the AB in the BAA is specific for CD3 and at least one other AB is a binding partner for any target listed in Table 9.
• AB2 of the BAA is specific for CD3 and AB1 is a binding partner for any target listed in Table 9.
• the unmasked EGFR-CD3 bispecific antibody exhibits EGFR-dependent tumor cell killing, while the doubly-masked EGFR-CD3 BAA reduces target-dependent cytotoxicity by more than 100,000-fold.
• BAAs potently induce tumor regressions.
• the maximum tolerated dose (MTD) of the EGFR-CD3 BAA is more than 60-fold higher than the MTD of the unmasked bispecific antibody, and the tolerated exposure (AUC) is more than 10,000-fold higher.
• BAAs have the potential to expand clinical opportunities for T cell-engaging bispecific therapies that are limited by on target toxicities, especially in solid tumors.
• an EGFR-CD3 BAA has the potential to address EGFR-expressing tumors that are poorly responsive to existing EGFR-directed therapies.
• Both the monospecific AAs and the BA As of the disclosure comprise at least one CM, when masked and not activated.
• the c!eavable moiety (CM) of the AA or BAA includes an amino acid sequence that can serve as a substrate for at least one protease, usually an extracellular protease.
• the CM may be selected based on a protease that is co-localized in tissue with the desired target of at least one AB of the BAA or AB of the AA.
• a CM can serve as a substrate for multiple proteases, e.g., a substrate for a serine protease and a second different protease, e.g. an MMP).
• a CM can serve as a substrate for more than one serine protease, e.g., a matriptase and a uPA.
• a CM can serve as a substrate for more than one MMP, e.g , an MMP9 and an MMP 14.
• a target of interest is co localized with a protease, where the substrate of the protease is known in the art.
• the target tissue can be a cancerous tissue, particularly cancerous tissue of a solid tumor.
• Non-limiting examples of disease include: all types of cancers, (such as, but not limited to breast, lung, colorectal, gastric, glioblastoma, ovarian, endometrial, renal, sarcoma, skin cancer, cervical, liver, bladder, cholangiocarcinoma, prostate, melanomas, head and neck cancer (e.g.. head and neck squamous cell cancer, pancreatic, etc.), rheumatoid arthritis, Crohn’s disease, SLE, cardiovascular damage, ischemia, etc.
• cancers such as, but not limited to breast, lung, colorectal, gastric, glioblastoma, ovarian, endometrial, renal, sarcoma, skin cancer, cervical, liver, bladder, cholangiocarcinoma, prostate, melanomas, head and neck cancer (e.g.. head and neck squamous cell cancer, pancreatic, etc.), rheumatoid arthritis, Crohn’
• indications would include leukemias, including T-cell acute lymphoblastic leukemia (T-ALL), lymphoblastic diseases including multiple myeloma, and solid tumors, including lung, colorectal, prostate, pancreatic and breast, including triple negative breast cancer.
• T-ALL T-cell acute lymphoblastic leukemia
• lymphoblastic diseases including multiple myeloma
• solid tumors including lung, colorectal, prostate, pancreatic and breast, including triple negative breast cancer.
• indications include bone disease or metastasis in cancer, regardless of primary tumor origin; breast cancer, including by way of non-limiting example, ER/PR+ breast cancer, Her2+ breast cancer, triple-negative breast cancer; colorectal cancer; endometrial cancer; gastric cancer; glioblastoma; head and neck cancer, such as head and neck squamous cell cancer; esophageal cancer; lung cancer, such as by way of non limiting example, non-small cell lung cancer; multiple myeloma ovarian cancer; pancreatic cancer; prostate cancer; sarcoma, such as osteosarcoma; renal cancer, such as by way of non- limiting example, renal cell carcinoma; and/or skin cancer, such as by way of non-limiting example, squamous cell cancer, basal cell carcinoma, or melanoma
• the CM is specifically cleaved by an enzyme at a rate of about 0.001 -1500 x 10 4 M or at least 0.001, 0 005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500 x 10 4 M ⁇ S 1 .
• contact between the enzyme and CM is made.
• the A A or BAA comprises at least a first AB coupled to a MM and a C
• the A A or BAA comprises an AB coupled to a MM via a CM
• the CM can he cleaved.
• Sufficient enzyme activity can refer to the ability of the enzyme to make contact with the CM and effect cleavage. It can readily be envisioned that an enzyme may be in the vicinity of the CM but is unable to cleave because of other cellular factors or protein modification of the enzyme.
• the CM has a length of up to 15 amino acids, a length of up to 20 amino acids, a length of up to 25 amino acids, a length of up to 30 amino acids, a length of up to 35 amino acids, a length of up to 40 amino acids, a length of up to 45 amino acids, a length of up to 50 amino acids, a length of up to 60 amino acids, a length in the range of 10-60 amino acids, a length in the range of 15-60 amino acids, a length in the range of 20-60 amino acids, a length in the range of 25-60 amino acids, a length in the range of 30-60 amino acids, a length in the range of 35-60 amino acids, a length in the range of 40-50 amino acids, a length in the range of 45-60 amino acids, a length in the range of 10-40 amino acids, a length in the range of 15- 40 amino acids, a length in the range of 20-40 amino acids, a length of 15- 40 amino acids, a length in the range of 20-40 amino acids, a length in the
• the AAs and BAAs of the disclosure comprise a prodomain, which comprises a MM.
• the MM is selected for use with a specific antibody or antibody fragment.
• the MM is not a natural binding partner of the AB. In some embodiments, the MM contains no or substantially no homology to any natural binding partner of the AB. In some embodiments the MM is no more than 5%, 10%, 15%, 20%,
• the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 50% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 25% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 20% identical to any natural binding partner of the AB. In some
• the MM is no more than 10% identical to any natural binding partner of the AB.
• Exemplary MMs of the di sclosure can have a length of up to 15 amino acids, a length of up to 20 amino acids, a length of up to 25 amino acids, a length of up to 30 amino acids, a length of up to 35 amino acids, a length of up to 40 amino acids, a length of up to 45 amino acids, a length of up to 50 amino acids, a length of up to 60 amino acids, a length in the range of 10-60 amino acids, a length in the range of 15-60 amino acids, a length in the range of 20-60 amino acids, a length in the range of 25-60 amino acids, a length in the range of 30-60 amino acids, a length in the range of 35-60 amino acids, a length in the range of 40- 50 amino acids, a length in the range of 45-60 amino acids, a length in the range of 10-40 amino acids, a length in the range of 15-40 amino acids, a length in the range of 20-40 amino acids, a length in the range of 25-40 amino acids, a
• linkers e.g., flexible linkers
• the AA/BAA eonstmcts so as to provide for flexibility at one or more of the MM-CM junction, the CM- AB/CM-scFv junction, or both.
• the AB, MM, and/or CM! may not contain a sufficient number of residues (e.g, Gly, Ser, Asp, Asn, especially Gly and Ser) to provide the desired flexibility.
• residues e.g, Gly, Ser, Asp, Asn, especially Gly and Ser
• the ability of such BAA constructs to remain intact or be activated as disclosed herein may benefit from introduction of one or more amino acids to provide for a flexible linker.
• an A A comprises one of the foll owing formulae (where the formula below represents an amino acid sequence in either N- to C- terminal direction or C- to N-terminal direction):
• MM, CM, and AB are as defined above; wherein LI and L2 are each independently and optionally present or absent, are the same or different flexible linkers that include at least 1 flexible amino acid (e.g., Gly, Ser).
• the BAA comprises 2 heavy chains, each comprising the structural arrangement from N-terminus to C-terminus of MM2-CM2-AB2-AB 1 HC and two light chains each comprising the structural arrangement from N-terminus to C-terminus of MM1-CM1-AB1 LC.
• the structure including with linkers is provided in FIG. 4.
• (MM2)-Ll-(CM2)-L2-(AB2) is linked to the heavy chain of AB1 and AB2 is a scFv
• Linkers suitable for use in compositions described herein are generally ones that provide flexibility of the modified AB or the AAs to facilitate the inhibition of the binding of the AB to the target. Such linkers are generally referred to as flexible linkers.
• Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.
• a suitable linker can be from 4 to 25 amino acids in length. In some embodiments, a suitable linker can be from 5 to 25 amino acids in length. In some embodiments, a suitable linker can be from 4 to 20 amino acids in length. In some embodiments, a suitable linker can be from 5 to 20 amino acids in length
• Exemplar ⁇ ' linkers include glycine polymers (G)n, glycine-serine polymers
• n is an integer of at least one
• Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between components. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev Computational Chem. 11173-142 (1992)). Exemplary linkers are provided in Table 9-1.
• design of an AA can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure to provide for a desired AA structure.
• the antibodies, or ABs of the AAs, and BA As are conjugated to an agent.
• the agent is a therapeutic agent.
• the agent is a detectable moiety.
• the agent is an antineoplastic agent.
• the agent is a toxin or fragment thereof.
• the agent is conjugated to the AB via a linker.
• the linker is a non- cleavable linker.
• the agent is a microtubule inhibitor.
• the agent is a nucleic acid damaging agent, such as a DNA alkylator or DNA intercalator, or other DNA damaging agent.
• the linker is a cleavable linker.
• the agent is an agent selected from the group listed in Table 10 Exemplary Pharmaceutical Agents tor Conjugation
• the antibody, AA or BAA comprises a detectable moiety.
• the detectable moiety is a diagnostic agent.
• the antibody, AA or BAA contains one or more disulfide bonds. In some embodiments, the antibody, AA or BAA contains one or more lysines. In some embodiments, the antibody, AA or BAA can be engineered to include one or more disulfide bonds or can be otherwise engineered to enable site-specific conjugation.
• the disclosure also provides an isolated nucleic acid molecule encoding an antibody, AA or BAA described herein, as well as vectors that include these isolated nucleic acid sequences.
• the disclosure provides methods of producing an antibody, AA or BAA by culturing a cell under conditions that lead to expression of the antibody, AA or BAA, wherein the cell comprises such a nucleic acid molecule.
• the cell comprises such a vector.
• the vector is pLW307.
• the vector is pLW291.
• the vector is pEF1049.
• the vector is pEF 105(3.
• the vector is pEFl 107.
• the vector is pEF1052. (these vectors are described and sequences provided below in Example 1)
• the antibodies/bi specific antibodies/ AAs/B AAs thereof may be used as therapeutic agents. Such agents will generally be employed to treat, alleviate, and/or prevent a disease or pathology in a subject.
• a therapeutic regimen is carried out by identifying a subject, e.g., a human patient or other mammal suffering from (or at risk of developing) a disorder using standard methods.
• Administration of the antibodies/bi specific antibodies/ AAs/BAAs thereof may abrogate or inhibit or interfere with the signal ing function of one or more of the targets.
• formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LipofectinTM), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P.
• alleviation or treatment of a disease or disorder involves the lessening of one or more symptoms or medical problems associated with the disease or disorder.
• the therapeutically effective amount of the drug can accomplish one or a combination of the following: reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., to decrease to some extent and/or stop) cancer cell infiltration into peripheral organs; inhibit tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer.
• a composition of this disclosure can be used to prevent the onset or
• a subject e.g., a human or other mammal, such as a non-human primate, companion animal (e.g., cat, dog, horse), farm animal, work animal, or zoo animal.
• companion animal e.g., cat, dog, horse
• farm animal e.g., work animal, or zoo animal.
• zoo animal e.g., a non-human primate, companion animal (e.g., cat, dog, horse), farm animal, work animal, or zoo animal.
• subject and patient are used interchangeably herein.
• a therapeutically effective amount of antihodies/hispecific antibodies/ AAs/BAAs thereof of the disclosure relates generally to the amount needed to achieve a therapeutic objective.
• Common ranges for therapeutically effective dosing of an antibodies/bispecific antibodies/ AAs/BAAs thereof of the disclosure may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Common dosing frequencies may range, for example, from twice daily to once a week
• Efficaciousness of treatment is determined in association with any known method for diagnosing or treating the particular disorder. Methods for the screening
• antibodies/bi specific antibodies/ AAs/BAAs that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA) and other immunologically mediated techniques known within the art
• antibodies/bispecific antibodies/A As/BA are used in methods known within the art relating to the localization and/or quantitation of the target (e.g., for use in measuring levels of one or more of the targets within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like).
• antibodies/bispecific antibodies/ AAs/BAAs are used to isolate one or more of the targets by standard techniques, such as immunoaffmity, chromatography or irnmunoprecipitation.
• An antibody, an AA, a bispecific antibody or a BAA can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
• detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
• suitable enzymes include horseradish peroxidase, alkaline phosphatase, b- ga!aetosidase, or acetylcholinesterase;
• suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
• suitable fluorescent materials include umbeliiferone, fluorescein, fluorescein isothiocyanate, rhodamine,
• an antibody, bispecific antibody, AA, BAA directed two or more targets can be used as an agent for detecting the presence of one or more of the targets (or a fragment thereof) in a sample.
• the antibody contains a detectable label.
• Antibodies are polyclonal, or in some embodiments, monoclonal. An intact antibody, or a fragment thereof (e.g, Fab, scFv, or Ffab’)?.) is used.
• the term“labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
• indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of an antibody with biotin such that it can be detected with fluorescently-labeled streptavidin.
• biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.
• biol ogi cal sample is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the disclosure can be used to detect a protein in a biological sample i vitro as well as in vivo.
• in vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELIS As), Western blots, immunoprecipitations, and immunofluorescence. Procedures for conducting immunoassays are described, for example in“ELISA: Theory and Practice:
• analyte protein in vivo techniques include introducing into a subject a l beled anti-analyte protein antibody.
• the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
• the antibodies, bispecifie antibodies, AAs, and bi specific antibodies of the disclosure are also useful in a variety of diagnostic and prophylactic formulations.
• an antibody, AA, bi specific antibody, BAA is administered to patients that are at risk of developing one or more of the aforementioned disorders.
• a patient’s or organ’s predisposition to one or more of the disorders can be determined using genotypic, serological or biochemical markers.
• BAA is administered to human individuals diagnosed with a clinical indication associated with one or more of the aforementioned disorders.
• an antibody, AA, bispecifie antibody, BAA is administered to mitigate or reverse the effects of the clinical indication.
• Antibodies, bispecifie antibodies, AAs, and bispecific antibodies are also useful in the detection of one or more targets in patient samples and accordingly are useful as diagnostics.
• the antibodies, bi specific antibodies, AAs, and bispecific antibodies of the disclosure are used in in vitro assays, e.g., ELISA, to detect one or more target levels in a patient sample.
• an antibody, AA, bispecific antibody, BAA is immobilized on a solid support (e.g, the well(s) of a microtiter plate).
• the immobilized antibody and/or AA serves as a capture antibody for any target(s) that may be present in a test sample.
• the solid support Prior to contacting the immobilized antibody/ AA with a patient sample, the solid support is rinsed and treated with a blocking agent such as milk protein or albumin to prevent nonspecific adsorption of the analyte.
• the wells are treated with a test sample suspected of containing the antigen, or with a solution containing a standard amount of the antigen.
• a test sample suspected of containing the antigen
• a solution containing a standard amount of the antigen is, e.g, a serum sample from a subject suspected of having levels of circulating antigen considered to be diagnostic of a pathology.
• the solid support is treated with a second antibody that is detectably labeled.
• the labeled second antibody serves as a detecting antibody.
• the level of detectable label is measured, and the concentration of target antigen(s) in the test sample is determined by comparison with a standard curve developed from the standard samples.
• Antibodies, bispecific antibodies, AAs, and BAAs can also be used in diagnostic and/or imaging methods.
• such methods are in vitro methods.
• such methods are in vivo methods.
• such methods are in situ methods.
• such methods are ex vivo methods.
• AAs, and bispecific antibodies having an enzymatically cleavable CM can be used to detect the presence or absence of an enzyme that is capable of cleaving the CM.
• Such AAs, and bi specific antibodies can be used in diagnostics, which can include in vivo detection (e.g., qualitative or quantitative) of enzyme activity (or, in some embodiments, an environment of increased reduction potential such as that which can provide for reduction of a disulfide bond) through measured accumulation of activated or bispecific activated antibodies (i.e., antibodies or bispecific antibodies resulting from cleavage of an AA or a BAA) in a given cell or tissue of a given host organism.
• activated or bispecific activated antibodies i.e., antibodies or bispecific antibodies resulting from cleavage of an AA or a BAA
• Such accumulation of activated bispecific antibodies indicates not only that the tissue expresses enzymatic activity (or an increased reduction potential depending on the nature of the CM) but also that the tissue expresses at least one target to which the activated bispecific antibody binds.
• the CM can be selected to be a protease substrate for a protease found at the site of a tumor, at the site of a viral or bacterial infection at a biologically confined site (e.g , such as in an abscess, in an organ, and the like), and the like.
• At least one of the AB can be one that binds a target antigen.
• a detectable label e.g., a fluorescent label or radioactive label or radiotracer
• Suitable detectable labels are discussed in the context of the above screening methods and additional specific examples are provided below.
• AAs will exhibit an increased rate of binding to disease tissue relative to tissues where the CM: specific enzyme is not present at a detectable level or is present at a lower level than in disease tissue or is inactive (e.g., in zymogen form or in complex with an inhibitor).
• antibodies, antibodies/bi specific antibodies/ AAs/BAAs of the present disclosure can be used to detect the presence or absence of a cleaving agent in a sample.
• the B AAs can be used to detect (either qualitatively or quantitatively) the presence of an enzyme in the sample.
• the antibodies/bispecific antibodies/ AAs/BAAs contain a CM susceptible to cleavage by reducing agent
• the antibodies/bispecific antibodies/AAs/BAAs can be used to detect (either qualitatively or quantitatively) the presence of reducing conditions in a sample.
• the antibodies/bispecific antibodies/AAs/BAAs can be delectably labeled, and can be bound to a support (e.g., a solid support., such as a slide or bead).
• a support e.g., a solid support., such as a slide or bead.
• the detectable label can be positioned on a portion of the antibodies/bispecific
• the detectable label can be a quenched fluorescent label or other label that is not detectable until cleavage has occurred.
• the assay can be conducted by, for example, contacting the immobilized, detectably labeled antibodies/bispecific antibodies/AAs/BAAs with a sample suspected of containing an enzyme and/or reducing agent for a time sufficient for cleavage to occur, then washing to remove excess sample and contaminants.
• the presence or absence of the cleaving agent (e.g, enzyme or reducing agent) in the sample is then assessed by a change in detectable signal of the antibodies/bispecific antibodies/AAs/BAAs prior to contacting with the sample e.g, the presence of and/or an increase in detectable signal due to cleavage of the antibodies/bispecific antibodies/AAs/BAAs by the cleaving agent in the sample
• Such detection methods can be adapted to also provide for detection of the presence or absence of a target that is capable of binding at least one AB of the antibodies/bispecific antibodies/AAs/BAAs of the present disclosure.
• the assays can be adapted to assess the presence or absence of a cleaving agent and the presence or absence of a target of interest.
• the presence or absence of the cleaving agent can be detected by the presence of and/or an increase in detectable label of the antibodies/bispecific antibodies/AAs/BAAs as described above, and the presence or absence of the target can be detected by detection of a target-AB complex e.g., by use of a detectably labeled anti -target antibody.
• AAs/BAAs of the present disclosure are also useful in in situ imaging for the validation of AA activation, e.g., by protease cleavage, and binding to a particular target.
• In situ imaging is a technique that enables localization of proteolytic activity and target in biological samples such as cell cultures or tissue sections. Using this technique, it is possible to confirm both binding to a given target and proteolytic activity based on the presence of a detectable label (e.g., a fluorescent label)
• a detectable label e.g., a fluorescent label
• an A A/BA A is labeled with a detectable label.
• the detectable label may be a fluorescent dye, (e.g. a fluorophore, Fluorescein Isothiocyanate (FITC), Rhodamine Isothiocyanate (TRITC), an Alexa Fluor® label), a near infrared (NIR) dye (e.g., Qdot® nanocrystals), a colloidal metal, a hapten, a radioactive marker, biotin and an amplification reagent such as streptavidin, or an enzyme (e.g. horseradish peroxidase or alkaline phosphatase).
• FITC Fluorescein Isothiocyanate
• TRITC Rhodamine Isothiocyanate
• Alexa Fluor® label Alexa Fluor® label
• NIR near infrared
• colloidal metal e.g., a hapten, a radioactive marker, biotin and
• Detection of the label in a sample that has been incubated with the labeled, AA or BAA indicates that the sample contains the target and contains a protease that is specific for the CM of the AAs or BAAs of the present disclosure.
• the presence of the protease can be confirmed using broad spectrum protease inhibitors such as those described herein, and/or by using an agent that is specific for the protease, for example, an antibody such as A! 1, which is specific for the protease matriptase (MT-SP1) and inhibits the proteolytic activity of MT-SP1; see e.g., International Publication Number WO 2010/129609, published 11 November 2010.
• the same approach of using broad spectrum protease inhibitors such as those described herein, and/or by using a more selective inhibitory ' agent can be used to identify a protease or class of proteases specific for the CM of the AAs or BAAs of the present disclosure.
• the presence of the target can be confirmed using an agent that is specific for the target or the detectable label can be competed with unlabeled target.
• unlabeled AA could be used, with detection by a labeled secondary' antibody or more complex detection system.
• Similar techniques are also useful for in vivo imaging where detection of the fluorescent signal in a subject, e.g, a mammal, including a human, indicates that the disease site contains the target and contains a protease that is specific for the CM: of the A As or BAAs of the present disclosure.
• formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LipofectinTM), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various mol ecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P.
• the antibodies, bispecific antibodies, AAs, or BAAs are administered in conjunction with one or more additional agents, or with a combination of additional agents.
• additional agents include current pharmaceutical and/or surgical therapies for an intended application. For example, they can be used in conjunction with an additional chemotherapeutic or anti- neoplastic agent.
• the antibodies, bi specific antibodies, AAs, or BAAs (or conjugated compositions thereof) of the present disclosure are administered in conjunction with one or more additional agents selected from the group consisting of antibodies, conjugated antibodies, AAs, conjugated AAs, bispecific antibodies, conjugated bi specific antibodies, BAAs, or conjugated BAAs.
• the antibody portion of any of the above-referenced additional agents is directed against a target such as one or more of the targets disclosed in Table 9. It is appreciated that in some embodiments the antibody portion of antibodies, bispecific antibodies, AAs, or BAAs (or conjugated compositions thereof) of the present disclosure and the antibody portion of the additional agent is directed against the same target (e.g. both may target EGFR).
• they are directed against the same target, but target different epitopes. In some embodiments, they are directed against different targets entirely (e.g., an activatable antibody of the present disclosure that targets EGFR may be administered in conjunction with an AA targeting a different target, likewise e.g. a BAA of the present disclosure that targets EGFR and CDS may be
• antibodies, bispecific antibodies, AAs or BAAs (or conjugated compositions thereof) of the disclosure are administered in conjunction with an immunotherapeutic agent.
• antibodies, bispecific antibodies, AAs or BAAs (or conjugated compositions thereof) of the disclosure are administered in conjunction with a chemotherapeutic agent.
• antibodies, bi specific antibodies, AAs or BAAs (or conjugated compositions thereof) of the disclosure are administered in conjunction with both an immunotherapeutic agent and a chemotherapeutic agent.
• one or more additional agents is administered with any of these combination embodiments.
• they are formulated into a single therapeutic composition, and the antibodies/bispecific antibodies/ AAs/BAAs thereof and the additional agent are administered simultaneously.
• the antibodies/bispecific antibodies/ AAs/BAAs thereof are administered separate from each other, e.g., each is formulated into a separate therapeutic composition, and the antibodies/bi specific antibodies/ AAs/BAAs thereof and the additional agent are administered simultaneously, or the antibodies/bispecific
• antibodies/A As/BA As thereof and the additional agent are administered at different times during a treatment regimen.
• the antibodies/bispecifie antibodies/ AAs/BAAs thereof and the additional agent can be administered in multiple doses.
• the antibodies/bispecific antibodies/ AAs/BAAs thereof can be incorporated into pharmaceutical compositions suitable for administration. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington’s Pharmaceutical Sciences: The Science And Practice Of Pharmacy 19th ed (Alfonso R. Gennaro, et ah, editors) Mack Pub. Co., Easton, Pa. ;
• compositions typically comprise the antibodies/bi specific
• antibodies/ AAs/BAAs thereof and a pharmaceutically acceptable carrier are provided.
• the term“pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington’s Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Suitable examples of such carriers or diluents include, but are not limited to, water, saline, ringer’s solutions, dextrose solution, and 5% human serum albumin.
• Liposomes and non-aqueous vehicles such as fixed oils may also be used.
• the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated.
• formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
• a pharmaceutical composition of the disclosure is formul ated to be compatible with its intended route of administration.
• routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration.
• Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethyienediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
• the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
• the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
• compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
• the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteri a and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
• the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
• Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
• Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients fro those enumerated above.
• methods of preparation are vacuum drying and freeze-drying that yi elds a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wiierein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
• the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin, or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
• a binder such as microcrystalline cellulose, gum tragacanth or gelatin
• an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch
• a lubricant such as magnesium stearate or Sterotes
• a glidant such as colloidal silicon dioxide
• the compounds are delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
• a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
• Systemic administration can also be by transmucosal or transdermal means.
• penetrants appropriate to the barrier to be permeated are used in the formulation.
• penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
• Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
• the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
• the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
• suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
• retention enemas for rectal delivery.
• the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as sustained/controlled release formulations, including implants and microencapsulated delivery systems.
• Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, poly glycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
• the active ingredients can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example,
• microcapsules respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in
• sustained-release preparations can be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g, films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2 ⁇ hydroxyethyl -methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
• copolymers ofL-glutamic acid and y ethyl-L-glutamate copolymers ofL-glutamic acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT 1M (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene- vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
• the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) and can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,81 1.
• Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• the specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteri stics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
• compositions can be included in a container, pack, or dispenser together with instructions for administration.
• the formulation can also contain more than one active compound as necessary for the particular indication being treated, for example, those with complementary ' activities that do not adversely affect each other.
• the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
• cytotoxic agent such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
• Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
• the active compounds are administered in combination therapy, i.e., combined with other agents, e.g., therapeutic agents, that are useful for treating pathological conditions or disorders, such as autoimmune disorders and inflammatory diseases.
• agents e.g., therapeutic agents
• the term“in combination” in this context means that the agents are given substantially contemporaneously, either simultaneously or sequentially. If given sequentially, at the onset of administration of the second compound, the first of the two compounds is still detectable at effective concentrations at the site of treatment.
• the combination therapy can include one or more antibodies/bispecific antibodies/ AAs/BAAs thereof of the disclosure coformulated with, and/or coadministered with, one or more additional therapeutic agents, e.g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and/or cytotoxic or cytostatic agents, as described in more detail below .
• additional therapeutic agents e.g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and/or cytotoxic or cytostatic agents, as described in more detail below .
• one or more antibodies/bispecific antibodies/ AAs/BAAs thereof described herein may be used in combination with two or more of the therapeutic agents described herein (e.g. one BAA administered with another BAA or AA of the disclosure, and the like).
• Such combinati on therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxi cities or complications associated with the various monotherapies.
• one or more antibodies of the disclosure can be coformulated with, and/or coadministered with, one or more anti-inflammatory drugs,
• Nonlimiting examples of the drugs or inhibitors that can be used in combination with the antibodies described herein include, but are not limited to, one or more of: nonsteroidal anti-inflammatory drug(s) (NSAIDs), e.g ., ibuprofen, tenidap, naproxen, meloxicam, piroxicam, diclofenac, and indomethacin; sulfasalazine; corticosteroids such as prednisolone; cytokine suppressive anti inflammatory drug(s) (CSAIDs); inhibitors of nucleotide biosynthesis, e.g.
• NSAIDs nonsteroidal anti-inflammatory drug(s)
• CSAIDs cytokine suppressive anti inflammatory drug(s)
• inhibitors of purine biosynthesis include NSAIDs, CSAIDs, (DHODH) inhibitors (e.g, ieflunomide), and folate antagonists (e.g, methotrexate).
• folate antagonists e.g., methotrexate (N-[4-[[(2,4-diamino-6- pteridinyBmethyl] methyl amino] benzoyl]-L-glutamic acid
• DHODH dihydroorotate dehydrogenase
• Suitable therapeutic agents for use in combination with the antibodies of the disclosure include NSAIDs, CSAIDs, (DHODH) inhibitors (e.g, ieflunomide), and folate antagonists (e.g, methotrexate).
• additional inhibitors include one or more of: corticosteroids (oral, inhaled and local injection); immunosuppressants, e.g, cyclosporin, tacrolimus (FK-506); and niTOR inhibitors, e.g., sirolimus (rapamycin - RAPAMUNE 1 ⁇ 1 or rapamycin derivatives, e.g, soluble rapamycin derivatives (e.g, ester rapamycin derivatives, e.g, CCI-779); agents that interfere with signaling by proinflammatory cytokines such as TNFa or IL-I (e.g.
• corticosteroids oral, inhaled and local injection
• immunosuppressants e.g, cyclosporin, tacrolimus (FK-506)
• niTOR inhibitors e.g., sirolimus (rapamycin - RAPAMUNE 1 ⁇ 1 or rapamycin derivatives, e.g, soluble rapamycin derivatives (e.
• IRAK, NIK, IKK, p38 or MAP kinase inhibitors COX2 inhibitors, e.g, celecoxib, rofecoxib, and variants thereof; phosphodiesterase inhibitors, e.g., R973401 (phosphodiesterase Type IV inhibitor); phospholipase inhi bitors, e.g., inhibitors of cytosolic phospholipase 2 (cPLA2) (e.g., trifluoromethyJ ketone analogs ); inhibitors of vascular endothelial cell growth factor or growth factor receptor, e.g., VEGF inhibitor and/or VEGF-R inhibitor; and inhibitors of angiogenesis.
• COX2 inhibitors e.g, celecoxib, rofecoxib, and variants thereof
• phosphodiesterase inhibitors e.g., R973401 (phosphodiesterase Type IV inhibitor)
• Suitable therapeutic agents for use in combination with the antibodies of the disclosure are immunosuppressants, e.g., cyclosporin, tacrolimus (FK-506); mTQR inhibitors, e.g, sirolimus (rapamycin) or rapamycin derivatives, e.g. , soluble rapamycin derivatives (e.g, ester rapamycin derivatives, e.g, CCI-779); CGX2 inhibitors, e.g, celecoxih and variants thereof, and phospholipase inhibitors, e.g, inhibitors of cytosolic phospholipase 2 (cPLA2), e.g, trifluoromethyl ketone analogs.
• immunosuppressants e.g., cyclosporin, tacrolimus (FK-506)
• mTQR inhibitors e.g, sirolimus (rapamycin) or rapamycin derivatives, e.g. , soluble rap
• Additional examples of therapeutic agents that can be combined with an antibody of the disclosure include one or more of: 6-mercaptopurines (6-MP); azathioprine
• sulphasalazine mesalazine; olsalazine; chloroquine/ hydroxychloroquine (PLAQUENIL ® ); pencill amine; aurothiomalate (intramuscular and oral); azathioprine; colchicine; beta-2 adrenoreceptor agonists (salbutamol, terbutaline, salmeteral); xanthines (theophylline, aminophylline); cromoglycate; nedocromil; ketotifen; ipratropium and oxitropium;
• mycophenolate mofetil mycophenolate mofetil; adenosine agonists; antithrombotic agents; complement inhibitors; and adrenergic agents.
• antibodies/bispecific antibodies/ AAs/BAAs thereof of the disclosure can be combined with one or more antibodies/bispecific antibodies/ AAs/BAAs thereof.
• kits comprising any one or more of the antibodies, A As, bispecific antibodies, and BAAs provided herein.
• the kit may comprise any one or more of the antibodies, AAs, bispecific antibodies, and BAAs provided herein in a composition suitable for storage or shipping.
• the kit may comprise a vessel, a diluent, a solvent, a second composition, or any component suitable for converting a storage or shipping composition in to a composition suitable for use in a method disclosed herein; the method may be, for instance, a therapeutic method disclosed herein.
• the kit may comprise instructions for use.
• Embodiment 1 A bispeeific activatabfe antibody (BAA), wherein said BAA, when activated, specifically binds to two targets, and wherein said BAA, when not activated, comprises the following structure:
• an IgG antibody that specifically binds to a first target wherein the AB 1 comprises:
• first prodomains each comprising a first masking moiety (MM1) linked to a first cleavable moiety (CM1) in the N-terminai to C-terminai direction, wherein the carboxyl terminus of each first prodomain is linked to the amino terminus of each light chain of the AB 1 , wherein
• the MMi reduces the binding of the AB1 to its target
• CM1 is a polypeptide that functions as a substrate for a first protease, b) two scFvs (AB2) that each specifically binds CD3r, wherein each AB2 comprises:
• VL light chain variable region
• VH heavy chain variable region
• VL comprises an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO:4 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 3;
• VL is linked to the VH by a linker L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 98 and SEQ ID NO: 108; and
• each second prodomain comprising a second masking moiety (MM2) linked to a second cleavable moiety (CM2) in the N-terminal to C-terminal direction, wherein the carboxyl terminus of each second prodomain is linked to the amino terminus of each AB2 wherein
• MM2 masking moiety
• CM2 second cleavable moiety
• the MM2 reduces the binding of the AB2 to CD 3 e;
• the MM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 105, SEQ ID NO: 106 and SEQ ID NO: 107; and
• CM2 is a polypeptide that functions as a substrate for a second protease
• Embodiment 2 The BAA of embodiment 1, wherein the VL is linked to the VH by a linker comprising amino acid sequence SEQ ID NO: 98
• Embodiment 3 The BAA of embodiment I, wherein the VL is linked to the VH by a linker comprising amino acid sequence SEQ ID NO: 108.
• Embodiment 4 The BAA of any one of embodiments 1 to 3, wherein the MM2 comprises amino acid sequence SEQ ID NO: 12.
• Embodiment 5 The BAA of any one of embodiments 1 to 3, wherein the MM2 comprises amino acid sequence SEQ ID NO: 105.
• Embodiment 6 The BAA of any one of embodiments 1 to 3, wherein the MM2 comprises amino acid sequence SEQ ID NO: 106.
• Embodiment 7 The BAA of any one of embodiments I to 3, wherein the MM2 comprises amino acid sequence SEQ ID NO: 107.
• Embodiment 8 The BAA of any one of embodiments 1 to 7, wherein the AB1 binds a tumor target.
• Embodiment 9 The BAA of any one of embodiments 1 to 8, wherein the AB l binds EGFR.
• Embodiment 10 The BAA of any one of embodiments I to 9, wherein the MM I comprises an amino acid sequence selected from the group consisting of sequences presented in Table 7 or Table 8.
• Embodiment 11 The BAA of any one of embodiments 1 to 10, wherein the
• MM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 78 and SEQ ID NO: 85.
• Embodiment 12 The BAA of any one of embodiments 1 to 11, wherein the MM1 comprises amino acid sequence SEQ ID NO: 78.
• Embodiment 13 The BAA of any one of embodiments 1 to 1 1, wherein the M T! comprises amino acid sequence SEQ ID NO: 85.
• Embodiment 14 The BAA of any one of embodiments 1 to 13, wherein the CM I or CM2 comprises the amino acid sequence of any one of the CMs listed in Table 4 or Table 4-1.
• Embodiment 15 The BAA of any one of embodiments 1 to 14, wherein the CM! or CM2 comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 17.
• Embodiment 16 The BAA of any one of embodiments ! to 15, wherein the CM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 16.
• Embodiment 17 The BAA of any one of embodiments 1 to 15, wherein the CM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 17.
• Embodiment 18 The BAA of any one of embodiments 1 to 17, wherein the AB1 comprises an Fc region comprising an amino acid substitution in at least one of amino acid positions L234, L235, N297, and P331 , as numbered by the EU index as set forth in Kabat, such that the BAA has reduced effector function.
• Embodiment 19 The BAA of embodiment! 8, wherein the AB1 comprises amino acid substitutions in at least two of amino acid positions L234, L235, and P331.
• Embodiment 20 The BAA of any one of embodiments 18 to 19, wherein the AB1 comprises amino acid substitutions at amino acid positions L234, L235, and P33 E
• Embodiment 21 The BAA of any one of embodiments 18 to 20, wherein the
• AB1 comprises L234F, L235E, and P331 S amino acid substitutions.
• Embodiment 22 The BAA of any one of embodiments 1 to 21, wherein the AB 1 comprises an Fc region comprising an amino acid substitution at N297
• Embodiment 23 The BAA of any one of embodiments 18 to 22, wherein the AB1 comprises L234F, L235E, P331S, and N297Q amino acid substitutions.
• Embodiment 24 The BAA of any one of embodiments 18 to 23, wherein the heavy chain of AB1 comprises an amino acid sequence selected from the group consisting SEQ ID NO: 115, SEQ ID NO: 1 16, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121 and SEQ ID NO: 122, as set forth in Table 4-2.
• Embodiment 25 The BAA of any one of embodiments 18 to 23, wherein the heavy chain of AB1 comprises an amino acid sequence selected from the group consisting SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, as set forth in Table 6.
• Embodiment 26 The BAA of any one of embodiments 1 to 7, and 10 to 25, wherein the first target is selected from the group consisting of the targets presented in Table 9.
• Embodiment 27 The BAA of any one of embodiments 1 to 26, wherein each of the first and second prodomains comprises a linker LI and a linker L2 according to the formula (MM)-L1-(CM)-L2 in the N-terminal to C-terminal direction.
• Embodiment 28 The BAA 0138, wherein the BAA comprises a heavy chain amino acid sequence as set forth in SEQ ID NO: 155 or SEQ ID NO: 124 and a light chain amino acid sequence as set forth in SEQ ID NO: 132 or SEQ ID NO: 140
• Embodiment 29 The BAA Cl 139, wherein the BAA comprises a heavy chain amino acid sequence as set forth in SEQ ID NO: 157 or SEQ ID NO: 126 and a light chain amino acid sequence as set forth in SEQ ID NO: 132 or SEQ ID NO: 140.
• Embodiment 30 The BAA 0158, wherein the BAA comprises a heavy chain amino acid sequence as set forth in SEQ ID NO: 161 or SEQ ID NO: 128 and a light chain amino acid sequence as set forth in SEQ ID NO: 132 or SEQ ID NO: 140.
• Embodiment 31 The BAA 0140, wherein the BAA comprises a heavy chain amino acid sequence as set forth in SEQ ID NO: 159 or SEQ ID NO:25 and a light chain amino acid sequence as set forth in SEQ ID NO: 132 or SEQ ID NO: 140.
• Embodiment 32 An activatable antibody (AA), wherein said AA, when activated, specifically binds to a target, and wherein said AA, when not activated, comprises the following structure:
• VL light chain variable region
• VH heavy chain variable region
• MM masking moiety
• CM cleavable moiety
• Embodiment 33 The AA of embodiment 32, wherein the target of the scFv is CD3r.
• Embodiment 34 An activatable antibody (AA) wherein said AA, when activated, specifically binds to a target, and wherein said AA, when not activated, comprises the following structure:
• MM masking moiety
• CM cleavable moiety
• Embodiment 35 The AA of any one of embodiments 32 to 34, wherein the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13-I7.
• Embodiment 36 The AA of any one of embodiments 32 to 34, wherein the CM comprises a substrate c!eavable by a serine protease or an MMP.
• Embodiment 37 The AA of any one of embodiments 32 to 34, wherein the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18-56.
• Embodiment 38 The AA of any one of embodiments 32 to 34, wherein the protease is an MMP.
• Embodiment 39 The AA of any one of embodiments 32 to 34, wherein the protease is a serine protease.
• Embodiment 40 The AA of any one of embodiments 32 to 39, wherein the VL comprises an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 4 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 3.
• Embodiment 41 The A A of any one of embodiments 32 to 40, wherein the prodomain comprises a linker LI and a linker L2 according to the formula (MM)-Ll-(CM)- L2 in the N-terminal to C-terminal direction.
• Embodiment 42 The AA or BAA of any one of embodiments 1 to 41, wherein the antigen binding fragment thereof is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.
• Embodiment 43 The AA or BAA of any one of embodiments 1 to 42, wherein the antibody is a rodent antibody, a chimeric antibody, a humanized antibody, or a fully human monoclonal antibody.
• Embodiment 44 The AA of any one of embodiments 1 to 43, wherein the AA is part of a BAA.
• Embodiment 45 A pharmaceutical composition comprising the AA or BAA of any one of embodiments 1 to 44 and, optionally, a carrier.
• Embodiment 46 The pharmaceutical composition of embodiment 45 comprising an additional agent.
• Embodiment 47 The pharmaceutical composition of embodiment 46, wherein the additional agent is a therapeutic agent.
• Embodiment 48 An isolated nucleic acid molecule encoding the AA or BAA of any one of embodiments 1 to 44.
• Embodiment 49 A vector comprising an isolated nucleic acid molecule of embodiment 48.
• Embodiment 50 A vector comprising the nucleic acid sequence of pEFl 049 [0289] Embodiment 51. A vector comprising the nucleic acid sequence of pEFlOSQ.
• Embodiment 52 A vector comprising the nucleic acid sequence of pEFl 107 [0291]
• Embodiment 53 A vector comprising the nucleic acid sequence of pEF1052 [0292]
• Embodiment 54 A cell comprising any one of the vectors of any one of embodiments 50 to 53.
• Embodiment 55 A cell comprising pEF1049 and pLW246
• Embodiment 56 A cell comprising pEFlOSO and pLW246.
• Embodiment 57 A cell comprising pEFl 107 and pLW246
• Embodiment 58 A cell comprising pEF1052 and pLW246.
• Embodiment 59 A method of producing the AA or BAA of any one of embodiments 1 to 44 by culturing a cell under conditions that lead to expression of the AA or BAA, wherein the cell comprises a nucleic acid molecule of embodiment 48 or a vector of anv one of embodiments 49 to 53, or a cell as set forth in anv one of embodiments 54 to 58.
• Embodiment 60 A method of treating, alleviating a symptom of, or delaying the progression of a disorder or disease comprising administering a therapeutically effective amount of the A A or BAA of any one of embodiments 1 to 44, or the pharmaceutical composition of any one of embodiments 45 to 47 to a subject in need thereof.
• Embodiment 61 The method of embodiment 60, wherein the disorder or disease comprises disease cells expressing EGER.
• Embodiment 62 The method of any one of embodiments 60 or 61, wherein the disorder or disease is cancer
• Embodiment 63 The method of embodiment 62, wherein the cancer is bladder cancer, breast cancer, cervical cancer, cho!angiocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, sarcoma, squamous cell cancer, or skin cancer.
• the cancer is bladder cancer, breast cancer, cervical cancer, cho!angiocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, sarcoma, squamous cell cancer, or skin cancer.
• Embodiment 64 A method of inhibiting angiogenesis in a subject compri sing administering a therapeutically effective amount of the AA or BAA of any one of
• embodiments 1 to 44 or the pharmaceutical composition of any one of embodiments 45 to 47 to a subject in need thereof
• Embodiment 65 The method of any one of embodiments 60 to 64, wherein the method comprises administering an additional agent.
• Embodiment 66 The method of embodiment 65 wherein the additional agent is a therapeutic agent.
• Embodiment 67 A method of reducing damage to healthy tissue caused by an antibody binding to its target on healthy tissue as well as on diseased tissue, the method comprising administering to a subject in need thereof an AA, BAA or a pharmaceutical composition comprising an AA or BAA, wherein said AA or BAA is an AA or BAA of any one of the embodiments 1 to 44
• Embodiment 68 A method to improve tolerability ' ⁇ of an antibody treatment comprising administering to a subject in need thereof an AA, BAA or a pharmaceutical composition comprising an AA or BAA, wherein said AA or BAA is an AA or BAA of any one of the embodiments 1 to 44
• Embodiment 69 A method to recruit T cells to tumor tissue comprising administering to a subject in need thereof an AA or BAA or a pharmaceutical composition comprising an AA or BAA, wherein said AA or BAA is an AA or BAA of any one of the embodiments 1 to 44.
• Embodiment 70 An AA or BAA of any one of embodiments 1 to 44, or a pharmaceutical composition of any one of embodiments 45 to 47, for use in a method of treating, alleviating a symptom of, or delaying the progression of a disorder or disease.
• Embodiment 71 The AA, BAA, or pharmaceutical composition for use according to embodiment 70, wherein the disorder or disease comprises disease cells expressing EGFR.
• Embodiment 72 The AA, BAA, or pharmaceutical composition for use according to embodiment 70 or 71, wherein the disorder or disease is cancer.
• Embodiment 73 The AA, BAA, or pharmaceutical composition for use according to embodiment 72, wherein the cancer is bladder cancer, breast cancer, cervical cancer, choiangiocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, sarcoma, squamous cell cancer, or skin cancer.
• the cancer is bladder cancer, breast cancer, cervical cancer, choiangiocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, sarcoma, squamous cell cancer, or skin cancer.
• Embodiment 74 An AA or BAA of any one of embodiments 1 to 44, or a pharmaceutical composition of any one of embodiments 45 to 47, for use in a method of inhibiting angiogenesis in a subject.
• Embodiment 75 The AA, BAA, or pharmaceutical composition for use according to any one of embodiments 70 to 74, wherein the method comprises the use of an additional agent.
• Embodiment 76 The AA, BAA, or pharmaceutical composition for use according to embodiment 75 wherein the additional agent is a therapeutic agent
• Embodiment 77 An AA or BAA of any one of embodiments 1 to 44, or a pharmaceutical composition of any one of embodiments 45 to 47, for use in a method of reducing damage to healthy tissue caused by an antibody binding to its target on healthy tissue as well as on diseased tissue.
• Embodiment 78 An AA or BAA of any one of embodiments 1 to 44, or a pharmaceutical composition of any one of embodiments 45 to 47, for use in a method to improve tolerability of an antibody treatment.
• Embodiment 79 An AA or BAA of any one of embodiments 1 to 44, or a pharmaceutical composition of any one of embodiments 45 to 47, for use in a method to recruit T cells to tumor tissue.
• EXAMPLE 1 Sequences, Vector Construction and Expression of Antibodies, BAAs and Activated BAAs
• GGT C AAAGGCTTCT ATCCC AGC GAC ATCGCCGT GGAGT GGG AG AGC AAT GGGC A
• linker L3 links the light chain and heavy chain, wherein L3 is selected from SEQ ID NO: 98 and SEQ ID NO: 108 ⁇
• Heavy chain HV20 (SEQ ID NO: 3)
• linker L3 links the light chain and heavy chain, wherein L3 is selected from SEQ ID NO: 98 and SEQ ID NO: 108.
• Heavy chain HV20 (SEQ ID NO: 3)
• linker L3 links the light chain and heavy chain, wherein L3 is selected from SEQ ID NO: 98 and SEQ ID NO: 108.
• Anti-CD3 scFv varia t v26 Light chain LV19 (SEQ ID NO: 4)
• linker L3 links the light chain and heavy chain, wherein L3 is selected from SEQ ID NO: 98 and SEQ ID NO: 108.
• the heavy and light chains were cloned separately into a mammalian expression vector using standard molecular biology techniques. Briefly, DNA fragments encoding the region of interest were amplified with primers binding to the terminal ends. Overlapping fragments were combined and amplified with flanking primers as needed to build the entire desired region. DNA fragments were subsequently cloned into the expression vector using a commercially available homologous recombination kit (MCLabs, South San Francisco, CA). The mammalian expression vector is a modified version of cDNA lM 3.l(+) from Invitrogen with selection marker of G418 or hygromycin. Mutations were introduced using the
• BAAs Dually Masked BA As
• AAs and BAAs were expressed in mammalian cells using a standard transfection kit (Life Technologies, Grand Island, NY). Briefly, 293 cells were transfected with nucleic acids using a lipid-based system, following the manufacturer’s recommended protocol. AAs and dually masked BAAs were purified from cell-free supernatant using Protein A beads (GE, Piscataway, NT) and concentrated using standard buffer exchange columns (Millipore, Temecula, CA).
• Protein A beads GE, Piscataway, NT
• Dually masked bispecific activatable antibodies were expressed using transient transfection in Expl293TM cells (Thermo Fisher Scientific, Waltham, MA, Catalog A14635). Synthetic DNA sequences encoding the proteins and signal peptides were ordered from Integrated DNA Technologies and cloned into transient expression vectors containing the CMV promoter. Endotoxin-free plasmid DNA preparations were confirmed by DNA sequencing prior to use. Plasmid DNA was transiently transfected into Expi293TM cells using the manufacturer’s recommended protocol. Supernatants were harvested four days post- transfection and purified using Protein A chromatography. The monomeric population for each expressed protein was purified by size exclusion chromatography (SEC) using a preparative-scale Superdex 200 10/30 GL column (GE Healthcare Life Sciences, Catalog # 17517501) in lx PBS pH 7.2 running buffer.
• SEC size exclusion chromatography
• concentration was determined by analytical SEC method described above. As shown in FIG. 1, Cl 106 showed a steeper drop in percent monomer with concentration compared to Cl 138, 0139, or 0140. 0139 showed the least tendency for concentration-dependent aggregation, whereas 0138 and 0140 showed a similar tendency for concentration-dependent aggregation between that of 0106 and 0139. The concentration-dependent aggregation tendency of these proteins was followed further by storing the concentrated samples (in PBS) at 4°C for an additional 2-to-4 days. SEC results in Table 12 suggest that percent monomer continued to drop for 0106 (3% over 2 days), 0138 (4.5% over 4 days), and 0140 (4.3% over 4 days) but seemed to be stable for 0139 (0.3% over 4 days).
• Each variant was SEC -purified, concentrated with a centrifugal concentrator and analyzed by SEC-HPLC. Table 12.
• ELISA assays were used to confirm and evaluate the binding of the intact dually masked bispecific activatable antibodies for CD3e and EGFR ligands.
• the purified dually masked bispecific activatable antibodies were activated by incubation with recombinant human u-plasminogen activator / urokinase (R&D systems, cat# 1310-SE) for 24 h in PBS buffer with 6% sucrose at 37C, re-purified by protein A chromatography, and re-analyzed by SDS-PAGE and SEC,
• CD3r ELISA Human CD3-epsilon protein (Aero Biosystems, Newark, DE, Catalog CDE-H5223) at 2pg/ml in IxPBS pH 7.2 (Thermo Fisher Scientific, Waltham, MA Catalog 20012043) was coated overnight at 4°C on Nunc MaxiSorpTM 96 well plates (Thermo Fisher Scientific, Waltham, MACatalog 439454). Plates were washed with lx PBS pH 7.2, 0.05% Tween-20 using a plate washer, blocked with 200 pL/well of 1% BSA, 0.05% Tween 20 in 1 x PBS pH 7.2 for 1 h at room temperature.
• FIG. 2A shows that 0138, 0139, and 0140 behave similar to 0106 in both intact as well as activated forms.
• EGFR ELISA Human EGFR-Fc protein (R&D Biosciences 344-ER) at l pg/ml in IxPBS pH 7.2 (Thermo Fisher Scientific, Waltham, MA Catalog 20012043) was coated overnight at 4°C on Greiner Bio-OneTM 96 well plates (Greiner, Monroe, NC, Catalog 655061). Plates w ? ere washed with lx PBS pH 7.2, 0.05% Tw ' een-20 using a plate washer, then blocked with 200 pL/well of 1% BSA, 0.05% Tween 20 in lx PBS pH 7.2 for 1 h at room temperature.
• FIG. 2B shows that intact 0138, 0139, and 0140 have similar EGFR binding as 0106. After activation, the EC50 for 0138, 0139, and 0140 looks similar to that of 0106 but the maximal signal is lower.
• Biological activity of intact and activated bi specific activatable antibodies were assayed using cytotoxicity assays.
• Human PBMCs were purchased from AHCells (Alameda, CA) and co-cultured with EGFR expressing HT29-iuc2 cells (Perkin Elmer, Inc., Waltham, MA (formally Caliper Life Sciences, Inc.) at a ratio of 10: 1 in RPMI ⁇ 1640-r-glutamax supplemented with 5% heat inactivated human serum (Sigma, Catalog H3667). Titrations of intact and activated Cl 106, 0138, 0139, and 0140 bispecific activatable antibodies were tested.
• cytotoxicity was evaluated using the ONE-Glo lM Luciferase Assay System (Promega, Madison, WI Catalog E6130). Luminescence was measured on the Infinite® M200 Pro (Tecan Trading AG, Switzerland). Percent cytotoxicity was calculated and plotted in GraphPad PRISM with curve fit analysis. As shown in FIG. 3, 0138, 0139, and 0140 are similar to 0106 in intact as well as activated forms. EXAMPLE 5. Dually masked, bispecific, activatable antibodies of the embodiments induced regression of established HT29-!uc2 tnmors in mice
• bispecific activatable antibodies 0106 and 0139 targeting EGFR and CD3e were analyzed for the ability to induce regression or reduce growth of established HT-29-Luc2 xenograft tumors in human T-cell engrafted NSG mice.
• the human colon cancer cell line HT29-Iuc2 was obtained from Perkin Elmer, Inc., Waltham, MA (formerly Caliper Life Sciences, Inc.) and cultured according to established procedures. Purified, frozen human PBMCs were obtained from Hemacare, Inc , Van Nuys, CA. NSG TM (NOD.Cg-PrkdcscidU2rgf mlw i l /SzJ) mice were obtained from The Jackson Laboratories, Bar Harbor, ME.
• mice On day 0, each mouse was inoculated subcutaneously at the right flank with 2xl0 6 HT29-luc2 cells in 100 m ⁇ RPMI + Glutamax, serum-free medium. Previously frozen PBMCs from a single donor were administered (i.p.) on day 3 at a CD3+ T cell to tumor cell ratio of 1 :1. When tumor volumes reached 150 mm 3 (approximately day 12), mice were randomized, assigned to treatment groups and dosed i.v. according to Table 13 Tumor volume was measured twice weekly. As shown in FIG. 5, 0106 and 0139 have equivalent efficacy in this model.
• EXAMPLE 6 Binding of dually masked, bispecific, AAs to EGFR+ HT-29 cells and CD3s+ Jurkat cells [0335] To determine if the described EGFR and CD3s masking peptides could inhibit binding in the context of a dually masked, bispecific, AA, a flow cytometry-based binding assay was performed.
• HT-29-luc2 Caliper
• Jurkat Clone E6-1, ATCC, TIB-152 ceils were cultured in RPMI-1640+glutamax (Life Technologies, Catalog 72400-047), 10% Heat Inactivated- Fetal Bovine Serum (HI-FBS, Life Technologies, Catalog 10438-026).
• the following bispecific, activated antibody 0106 (act-TCB), and dually masked, bispecific, AAs 0106 and 0139 were tested.
• Two versions of the CD3 mask were utilized, namely the CD3 mask in 0106 versus the CD3 mask in 0139.
• HT29-luc2 cells were detached with VerseneTM (Life Technologies, Catalog 15040- 066), washed, plated in 96 well plates at 150,000 cells/well, and re-suspended in 50 pL of primary antibody Jurkat cells were counted and plated as described for HT29-luc2 cells. Titrations of primary antibody started at the concentrations indicated in FIG. 6 followed by 3- fold serial dilutions in FACS Stain Buffer + 2% FBS (BD Pharmingen, Catalog 554656).
• MFI median fluorescence intensity
• FIG. 6 depicts reduced binding to both EGFR and CD3 of intact activatable bispecific antibodies Cl 106 and 0139 relative to the activated bispecific antibody as represented by a right shift of the binding curves.
• EXAMPLE 7 Biological activity of dually masked bispecific activatable antibodies [0339] Biological activity of intact and activated bispecific activatable antibodies was assayed using cytotoxicity assays. Human PBMCs were purchased from Stemcell
• EGFR expressing cancer cell lines HT29 ⁇ luc2 (Perkin Elmer, Inc., Waltham, MA (formally Caliper Life Sciences, Inc.), NCI- N87 (ATCC), OE33 (ATCC), SKBR3 (ATCC), or SKOV3 (ATCC) at a ratio of 10:1 , 6: 1, 8: 1, 6: 1, or 6: 1 respectively in RPMI-1640+glutamax supplemented with 5% heat inactivated human serum (Sigma, Catalog H3667). Titrations of activated 0106 (act-TCB) and intact 0106 and 0139 bi specific activatable antibodies were tested.
• cytotoxicity was evaluated using the ONE-GloTM Luciferase Assay System (Promega, Madison, WI Catalog E6130). Luminescence was measured on the Infinite® M200 Pro (Tecan Trading AG, Switzerland). Percent cytotoxicity was calculated and plotted in GraphPad PRISM with curve fit analysi
• the intact bi specific activatable antibodies have a shifted dose response curve relative to the activated bispecific antibody, and 0139 activity is similar to 0106 in these assays.
• bispecific activatable antibodies 0106 and 0139 targeting EGFR and CD3s were analyzed for the ability to induce regression or reduce growth of established HOT 16 xenograft tumors in human T-cell engrafted NSG mice.
• the human colon cancer cell line HCT116 was obtained from ATCC and was cultured in RPMI + Glutamax + 10% FBS according to established procedures.
• the tumor model was carried out as described in Example 5. As shown in FIG 8, 0106 and 0139 have equivalent efficacy in this model.
• EXAMPLE 9 Pharmacokinetics of Dually masked BAAs in eynomolgos monkeys

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